Abstract Effect anti-HIV drugs have focused on a limited number of targets. While these drugs are currently highly efficacious there is a need to continue to develop new targets for two reasons. First, it is possible that over the course of treating infected people for the decades to come drug resistance could become a problem; thus developing new targets and inhibitors now provides the theoretical underpinning for the development of new drugs in the future. Second, inhibitors represent chemical probes that can complement genetic approaches in terms of studying biological phenomena. Thus inhibitors that are not drugs still have intrinsic value as tools in understanding complex biological systems. We have previously shown that the MA/CA protease cleavage site in the HIV-1 Gag protein must be cleaved to near completion to allow an infectious virus particle to form. Even 10% under-cleavage of this site poisons the assembly process, likely because some of the uncleaved MA/CA fusion protein participates in capsid formation and poisons this highly regulated process. Bevirimat formed a conceptual basis for thinking about inhibitors of specific cleavage sites, in this case CA/SP1. However, among all of the cleavage sites in Gag the MA/CA cleavage site is by far the most sensitive to under-processing to affect infectivity. For this reason we undertook to develop a biochemical assay that could be used in a high throughput screen for small molecule inhibitors. That screen (775,000 compounds screened by SRI) is now completed with the most active compounds having an IC50 starting at 1 uM. These compounds have already been shown not to be HIV-1 protease inhibitors. In this application we propose four aims. First, we will test the most active compounds in a secondary screen using our two-substrate gel-based assay that includes both the target MA/CA substrate and as an internal control a substrate that can be cleaved by the HIV-1 protease but which should not be a target of the inhibitor. Second, we will measure EC50s for viral replication using a novel assay that will greatly enhance our sensitivity to detect specific inhibition. Third, we will measure EC50s for a panel of transmitted/founder viruses and viruses from different clades to provide an initial assessment of the effect of sequence variability on sensitivity. And fourth, for those compounds that have a sufficient therapeutic index we will start to select for resistance to validate the MA/CA target. With this effort we will identify the most promising hits in this new screen that can then be used in the future as the basis for structural studies and SAR.